Because timely and accurate identification of members of the Pseudallescheria/ Scedosporium species complex (PSC) is clinically relevant, the objective of this investigation was to study the stability and influence of the main variable factors in the routine clinical laboratory to the potential use the Matrix-Assisted Laser Desorption Ionization-Time-Of-Flight (MALDI-TOF MS) in the identification of these fungi. Twenty-two PSC reference strains, three clinical isolates, an αHCCA matrix, and an Autoflex I spectrometer with BioTyper software (Bruker) were employed in this study. Intra-and inter-specimen composite correlation indices for each MS spectrum as compared to a reference spectrum were computed. MS identification was stable after the fungi were subcultured over a 1-month period. While neither culture medium (Sabouraud vs. Malt extract) nor protein extraction methods (formic acid vs. trifluoroacetic acid) significantly influenced the quality of the MS identifications, they were considerably increased from day 3 to day 6 of incubation. MALDI-TOF MS can be used in the routine clinical laboratory in the identification of members of the complex provided that valid spectra libraries are developed. Although preliminary results are encouraging, further studies are warranted to demonstrate whether MS can distinguish the species that have recently been described using multilocus sequence analysis within P. boydii sl. and to validate its use in the routine clinical laboratory for identifying clinically relevant moulds.